Compositions and methods for obtaining cells to treat heart tissue

ABSTRACT

This document relates to compositions containing cardiogenic factors, to methods to obtain cells by culturing initial cells in the presence of such factors; and methods of administering the obtained cells to heart tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/994,626, filed Jan. 11, 2011 (now U.S. Pat. No. 8,835,384), which isa National Stage application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2009/044714, having an international filing dateof May 20, 2009, which claims benefit of priority from InternationalApplication No. PCT/US2008/064895, filed on May 27, 2008. Thedisclosures of the prior applications are considered part of (and areincorporated by reference in) the disclosure of this application.

TECHNICAL FIELD

This document relates to methods and materials involved in obtaining andusing cardiac cells. For example, this document relates to methods andmaterials for providing mammalian heart tissue with cells (e.g.,differentiated cardioprogenitor cells or cardiopoietic cells) thatincorporate into the heart tissue as functional cardiomyocytes.

BACKGROUND INFORMATION

Cardiovascular disease is a leading cause of morbidity and mortalityworldwide, despite advances in patient management. In contrast totissues with high reparative capacity, heart tissue is vulnerable toirreparable damage. Cell-based regenerative cardiovascular medicine is,therefore, being pursued in the clinical setting.

The recent advent of stem cell biology extends the scope of currentmodels of practice from traditional palliation towards curative repair.Typically, clinical experience has been based on adult stem cellsrecruited from autologous sources and delivered in an unaltered state.First generation biologics are naïve human stem cells, identified asreadily accessible cytotypes. It has been shown that particularindividuals improve on delivery of naïve human stem cells.

DEFINITIONS

Within the frame of the present document, and unless indicated to thecontrary, the terms designated below between quotes have the followingdefinitions.

The term ‘hMSCs’ means human mesenchymal stem cells.

The ‘cardio-generative potential’ of a cell designates the ability ofthis cell to succeed to generate cardiac cells for instance myocardium,when injected into an infracted heart.

‘Cardiopoietic cells’ (CP) are cells engaged in the way ofdifferentiation from a non-differentiated cell. A ‘cardiopoietic cell’exhibits a cardiac differentiation defined by nuclear translocation ofthe early cardiac transcription factor Nkx2.5 and the late cardiactranscription factor MEF2C (Behfar et al. Derivation of a cardiopoieticpopulation from human mesenchymal stem yields progeny, Nature ClinicalPractice, Cardiovascular Medicine, March 2006 vol. 3 supplement 1, pagesS78-S82). Nuclear translocation of cardiac transcription factor GATA4can be observed. Cardiopoietic cells can lack sarcomeres and can lackexpression of sarcomeric proteins. A cardiopoietic cell keeps thecapacity to divide itself. Cardiopoietic cells are also called‘cardiomyocyte precursors’ or ‘cardiomyocyte progenitor cells’ becausethey may differentiate into cardiomyocytes. In the context of thepresent document, cardiopoietic cells may be derived from human adultmesenchymal stem cells (hMSCs). ‘CP-hMSCs’ designates such cardiopoieticcells derived from human adult mesenchymal stem cells.

A ‘cocktail’ or ‘cardiogenic cocktail’ designates a compositioncontaining at least two cardiogenic substances.

A ‘cardiogenic substance’ is a substance which improves thecardio-generative potential of a cell.

A ‘cocktail-guided cell’ or ‘cell guided towards cardiopoiesis’ is acell which has been put into contact with a cocktail and further entersinto differentiation.

The ‘differentiation’ is the process by which a less specialized cellbecomes a more specialized cell.

The ‘ejection fraction’ means the fraction of blood pumped out during aheart beat. Without a qualifier, the term ejection fraction refersspecifically to that of the left ventricle (left ventricular ejectionfraction or LVEF).

The ‘change of ejection fraction’ means the difference between ejectionfraction of the heart of an animal treated with cells injected into itsinfarcted heart, measured after a given time and the ejection fractionmeasured prior to injection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

SUMMARY

The invention relates to compositions comprising TGFβ-1, BMP4,α-thrombin, a compound selected from the group consisting ofCardiotrophin and IL-6, and a compound selected from the groupconsisting of Cardiogenol C and retinoic acid. In a preferredembodiment, the compositions of the invention comprise TGFβ-1, BMP4,α-thrombin, Cardiotrophin and Cardiogenol C. The compositions of theinvention may comprise at least one compound selected from the groupconsisting of FGF-2, IGF-1, Activin-A, TNF-α, FGF-4, LIF, VEGF-A andcombinations thereof. They may also comprise FGF-2, IGF-1 and Activin-A.Other preferred compositions of the invention comprises Activin-A,FGF-2, IL-6, IGF-1 and retinoic acid. According to an alternativeembodiment, the compositions of the invention can lack at least onecompound chosen in the group consisting of TNF-α, FGF-4, LIF, andVEGF-A.

When one of the following compounds is present in a composition of theinvention, it may be present in an amount of between 1 and 5 ng of saidTGFβ-1 per ml, between 1 and 10 ng of said BMP4 per ml, between 0.5 and5 ng of said Cardiotrophin per ml, between 0.5 and 5 units of saidα-thrombin per ml, and between 50 and 500 nM of said Cardiogenol C,between 1 and 10 ng of said FGF-2 per ml, between 10 and 100 ng of saidIGF-1 per ml, between 1 and 50 ng of said Activin-A per ml, between 1and 50 ng of said TNF-α per ml, between 1 and 20 ng of said FGF-4 perml, between 10 and 100 ng of said IL-6 per ml, between 1 and 10 units ofsaid LIF per ml, between 1 and 50 ng of said VEGF-A per ml, between 0.1and 1.0 μM of said retinoic acid per ml.

Preferred compositions according to the invention comprise recombinantTGFβ-1 (2.5 ng/ml), BMP4 (5 ng/ml), Cardiotrophin (1 ng/ml), CardiogenolC (100 nM), used in a combinatorial fashion. Particularly preferredcompositions of the invention comprises such compounds and furthercomprise α-thrombin, (1 U/ml), FGF-2 (10 ng/ml), IGF-1 (50 ng/ml) andActivin-A (5 ng/ml).

Other preferred compositions of the invention comprise recombinantTGFβ-1 (2.5 ng/ml), BMP4 (5 ng/ml), Activin-A (5 ng/ml), FGF-2 (10ng/ml), IL-6 (100 ng/ml), Factor-IIa (hα-thrombin, 1 U/ml), IGF-1 (50ng/ml), and retinoic acid (1 μM) used in a combinatorial fashion.

Preferably, the compositions of the invention are comprised in a mediumselected from the group consisting of media containing of foetal calfserum, human serum, platelet lysate, and mixtures thereof.

The invention also relates to a method for obtaining from initial cellsdifferentiated cells expressing an elevated level of at least one of themRNAs selected from the group consisting of MEF2c mRNA, MESP-1 mRNA,Tbx-5 mRNA, GATA4 mRNA, Flk-1 mRNA, GATA6 mRNA, Fog-1 mRNA, andcombinations thereof, and/or have at least one polypeptide selected fromthe group consisting of Nkx2.5 polypeptides, MEF2C polypeptides, Tbx-5polypeptides, FOG-2 polypeptides, GATA-4 polypeptides MESP-1polypeptides, and combinations thereof, wherein said at least onepolypeptide is associated with the nuclei of said differentiated cells,wherein said method comprises culturing initial cells in the presence ofa composition according to the invention In such methods, thedifferentiated cells express preferably an elevated level of MEF2c mRNAand MESP-1 mRNA.

In a preferred embodiment of the invention, the initial cells aremesenchymal stem cells. Such cells can be bone marrow-derived stemcells. They can express CD90, CD105, CD133, CD166, CD29, and CD44 on thecell surface and do not express CD14, CD34, and CD45 on the cellsurface.

Most preferably, the differentiated cells are cardiopoietic cells.

Another aspect of the invention is a method for deliveringdifferentiated cells to a mammal, wherein said method comprises:

(a) determining that a sample of cells from a population ofdifferentiated cells comprises cells that express an elevated level ofat least one of the mRNA selected from the group consisting of MEF2cmRNA, MESP-1 mRNA, Tbx-5 mRNA, GATA4 mRNA, Flk-1 mRNA, GATA6 mRNA, Fog-1mRNA, and combinations thereof, and/or have at least one polypeptideselected from the group consisting of Nkx2.5 polypeptides, MEF2Cpolypeptides, Tbx-5 polypeptides, MESP-1 polypeptides, GATA-4polypeptides, FOG-2 polypeptides, and combinations thereof, wherein saidpolypeptide is associated with the nuclei of said differentiated cells,and

(b) administering cells from said population of differentiated cells tosaid mammal. Said population of differentiated cells can be obtainedfrom said original cells cultured in the presence of any of saidcompositions according to the invention. In a particular embodiment,said step (a) may comprise using a reverse transcription polymerasechain reaction or using immunocytochemistry. Said administering stepcomprises may comprise administering said cells via an administrationselected from the group consisting of systemic, intracardiac, andintracoronary administrations.

Another aspect of the invention is a method for providing heart tissuewith cardiomyocytes, wherein said method comprises administering, tosaid heart tissue, said differentiated cells of claims obtainable bycontact with a composition according to the invention.

Details of one or more embodiments of the invention are set forth in theaccompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and from the claims.

DETAILED DESCRIPTION

This document provides methods and materials related to cardiac cells(e.g., differentiated cardioprogenitor cells). For example, thisdocument provides cells having the ability to incorporate into hearttissue as functional cardiomyocytes, methods for making such cells,compositions for making such cells, and methods for determining whetheror not a population of cells (e.g., differentiated cardioprogenitorcells) contains cells having the ability to incorporate into hearttissue as functional cardiomyocytes. This document also provides methodsand materials for providing heart tissue (e.g., human heart tissue) withfunctional cardiomyocytes.

The differentiated cardioprogenitor cells provided herein can be fromany species including, without limitation, humans, monkeys, horses,dogs, cats, rats, or mice. For example, differentiated cardioprogenitorcells can be mammalian (e.g., human) differentiated cardioprogenitorcells.

In some cases, differentiated cardioprogenitor cells provided hereinhave the ability to incorporate into heart tissue as functionalcardiomyocytes.

Any appropriate method can be used to obtain differentiatedcardioprogenitor cells. For example, differentiated cardioprogenitorcells can be derived from stem cells such as mammalian (e.g., human)stem cells.

In some cases, differentiated cardioprogenitor cells can be derived fromembryonic stem cells. In some cases, differentiated cardioprogenitorcells can be derived from mesenchymal stem cells. Mesenchymal stem cellscan be obtained from any source. For example, mesenchymal stem cells canbe obtained from mammalian (e.g., human) tissue such as bone marrow andtrabecular bone. Mesenchymal stem cells can be cultured in vitro. Forexample, mesenchymal stem cells can be expanded in number in vitro.Mesenchymal stem cells can express or not express a polypeptide markeron the cell surface. For example, mesenchymal stem cells can expressCD133, CD90, CD105, CD166, CD29, and CD44 on the cell surface and notexpress CD14, CD34, and CD45 on the cell surface.

Any appropriate method can be used to derive differentiatedcardioprogenitor cells from stem cells (e.g., mesenchymal stem cells).For example, differentiated cardioprogenitor cells can be derived frommesenchymal stem cells by incubating the mesenchymal stem cells with acomposition (e.g., culture media). The composition can be anyappropriate composition containing one or more factors. The factors canbe any type of factors such as polypeptides, steroids, hormones, andsmall molecules. Examples of such factors include, without limitation,TGFβ, BMP, FGF-2, IGF-1, Activin-A, Cardiotrophin, α-thrombin, andCardiogenol C.

In one embodiment, media containing TGFβ, BMP, Cardiotrophin,α-thrombin, and Cardiogenol C can be used to obtain differentiatedcardioprogenitor cells from stem cells (e.g., mesenchymal stem cells).In such cases, FGF-2, IGF-1, Activin-A, or a combination thereof can beadded to the medium after an initial culture period (e.g., one or twodays) with medium containing TGFβ, BMP, Cardiotrophin, α-thrombin, andCardiogenol C.

TGFβ can be any polypeptide having TGFβ activity, such as human TGFβ.For example, TGFβ can be recombinant TGFβ or synthetic TGFβ. In oneembodiment, TGFβ can be TGFβ-1. Any appropriate concentration of TGFβcan be used. For example, between 1 and 10 ng of TGF-β per ml (e.g.,about 2.5 ng of TGFβ1 per ml) can be used.

BMP can be any polypeptide having BMP activity, such as human BMP. Forexample, BMP can be recombinant BMP or synthetic BMP. In one embodiment,BMP can be BMP4. Any concentration of BMP can be used. For example,between 1 and 20 ng of BMP per ml (e.g., about 5 ng of BMP4 per ml) canbe used.

FGF-2 can be any polypeptide having FGF-2 activity, such as human FGF-2.For example, FGF-2 can be recombinant FGF-2 or synthetic FGF-2. Anyconcentration of FGF-2 can be used. For example, between 1 and 20 ng ofFGF-2 per ml (e.g., about 5 ng of FGF-2 per ml) can be used.

IGF-1 can be any polypeptide having IGF-1 activity, such as human IGF-1.For example, IGF-1 can be recombinant IGF-1 or synthetic IGF-1. Anyconcentration of IGF-1 can be used. For example, between 10 and 100 ngof IGF-1 per ml (e.g., about 50 ng of IGF-1 per ml) can be used.

Activin-A can be any polypeptide having Activin-A activity, such ashuman Activin-A. For example, Activin-A can be recombinant Activin-A orsynthetic Activin-A. Any concentration of Activin-A can be used. Forexample, between 1 and 50 ng of Activin-A per ml (e.g., about 10 ng ofActivin-A per ml) can be used.

α-Thrombin can be any polypeptide having α-thrombin activity, such ashuman α-thrombin. For example, α-thrombin can be recombinant α-thrombinor synthetic α-thrombin. Any concentration of α-thrombin can be used.For example, between 0.5 and 10 units of α-thrombin per ml (e.g., about1 unit of α-thrombin per ml) can be used.

Cardiotrophin can be any polypeptide having Cardiotrophin activity, suchas human Cardiotrophin-1. For example, Cardiotrophin can be recombinantCardiotrophin or synthetic Cardiotrophin. Any concentration ofCardiotrophin can be used. For example, between 0.5 and 10 ng ofCardiotrophin per ml (e.g., about 1 ng of Cardiotrophin-1 per ml) can beused.

IL-6 can be any polypeptide having IL-6 activity, such as human IL-6.For example, IL-6 can be recombinant IL-6 or synthetic IL-6. Anyconcentration of IL-6 can be used. For example, between 100 and 200 ngof IL-6 per ml can be used.

Any concentration of Cardiogenol C or a pharmaceutically acceptable saltthereof (e.g., Cardiogenol C hydrochloride) can be used. For example,between 10 and 1000 nM of Cardiogenol C (e.g., about 100 nM ofCardiogenol C) can be used.

Retinoic acid can be any molecule having retinoic acid activity, such assynthetic retinoic acid, natural retinoic acid, a vitamin A metabolite,a natural derivative of vitamin A, or a synthetic derivative of vitaminA. Any concentration of retinoic acid can be used. For example, between1×10⁻⁶ and 2×10⁻⁶ μM of retinoic acid can be used.

In some cases, serum-containing or serum-free media supplemented withTGFβ-1 (e.g., 2.5 ng/ml), BMP4 (e.g., 5 ng/ml), FGF-2 (e.g., 5 ng/ml),IGF-1 (e.g., 50 ng/ml), Activin-A (e.g., 10 ng/ml), Cardiotrophin (e.g.,1 ng/ml), α-thrombin (e.g., 1 Unit/ml), and Cardiogenol C (e.g., 100 nM)can be used to obtain differentiated cardioprogenitor cells from stemcells (e.g., mesenchymal stem cells). In some cases, the media (e.g.,serum-containing or serum-free media) can contain platelet lysate (e.g.,a human platelet lysate)

In some cases, the composition used to obtain differentiatedcardioprogenitor cells from mesenchymal stem cells can containadditional optional factors such as TNF-α, LIF, and VEGF-A.

TNF-α can be any polypeptide having TNF-α activity, such as human TNF-α.For example, TNF-α can be recombinant TNF-α or synthetic TNF-α. Anyconcentration of TNF-α can be used. For example, between 5 and 50 ng ofTNF-α per ml can be used.

LIF can be any polypeptide having LIF activity, such as human LIF. Forexample, LIF can be recombinant LIF or synthetic LIF. Any concentrationof LIF can be used. For example, between 2.5 and 100 ng of LIF per mlcan be used.

VEGF-A can be any polypeptide having VEGF-A activity, such as humanVEGF-A. For example, VEGF-A can be recombinant VEGF-A or syntheticVEGF-A. Any concentration of VEGF-A can be used. For example, between 5and 200 ng of VEGF-A per ml can be used.

A composition provided herein can contain any combination of factors.For example, a composition provided herein can contain TGFβ-1, BMP4,Activin-A, Cardiotrophin, α-thrombin, and Cardiogenol C. In some cases,a composition provided herein can contain TGFβ-1, BMP4, FGF-2, IGF-1,Cardiotrophin, α-thrombin, and Cardiogenol C. In some cases, acomposition provided herein can contain TGFβ-1, BMP4, FGF-2, IGF-1,Cardiotrophin, α-thrombin, and Cardiogenol C. In some cases, acomposition provided herein can lack TNF-α, IL-6, LIF, VEGF-A, retinoicacid, or any combination thereof (e.g., IL-6, LIF, VEGF-A, and retinoicacid; LIF, VEGF-A, and retinoic acid; or TNF-α, IL-6, LIF, and VEGF-A).

A composition provided herein can be prepared using any appropriatemethod. For example, a composition provided herein can be prepared usingcommercially available factors. In some cases, a composition providedherein can be prepared to contain cells lysates (e.g., a plateletlysate) or conditioned media from cells such as cardiomyocyte cells orTNF-α-stimulated endodermal cells. For example, a composition providedherein can be prepared using a platelet lysate supplemented withcommercially available factors. In some cases, a composition providedherein can be prepared using factors isolated from conditioned medium.In some cases, the factors can be dissolved in media such as cellculture media that does or does not contain serum.

Any appropriate method can be used to incubate stem cells (e.g.,mesenchymal stem cells) with a composition provided herein to obtaindifferentiated cardioprogenitor cells having the ability to incorporateinto heart tissue as functional cardiomyocytes. For example, mesenchymalstem cells can be incubated with a composition provided herein for 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40, 45, or 50 days. In some cases, a compositionprovided herein and used to incubate mesenchymal stem cells can bereplaced every day or every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 days.

In some cases, mesenchymal stem cells can be incubated with acomposition provided herein in the presence or absence of serum. Anyappropriate cell density can be used when incubating stem cells with acomposition provided herein. For example, between about 1000 and 2000mesenchymal stem cells per cm² (e.g., between about 1500-2000 cells/cm²)can be incubated with a composition provided herein to obtaindifferentiated cardioprogenitor cells.

Once stem cells (e.g., mesenchymal stem cells) have been incubated witha composition provided herein or otherwise treated with differentiationfactors, the state of differentiation can be monitored to determinewhether or not the stem cells differentiated into differentiatedcardioprogenitor cells having the ability to incorporate into hearttissue as functional cardiomyocytes. For example, a sample of cells canbe collected and assessed using techniques such as Western blotting,fluorescence-activated cell sorting (FACS), immunostaining, laserconfocal microscopy, and reverse transcription polymerase chain reaction(RT-PCR) techniques (e.g., quantitative RT-PCR). In some cases, cellsfound to express an elevated level of MEF2c, MESP-1, Tbx-5, Nkx2.5,GATA6, Flk-1, Fog 1 and Fog 2 polypeptides or mRNA can be selected foradministration into a mammal to treat heart tissue.

As described herein, differentiated cardioprogenitor cells derived frommesenchymal stem cells cultured with a platelet lysate containingTGFβ-1, BMP4, FGF-2, IGF-1, Activin-A, Cardiotrophin, α-thrombin, andCardiogenol C exhibited a 2 to 5-fold increase in MEF2c mRNA, MESP-1mRNA, Tbx-5 mRNA, GATA6 mRNA, Flk-1 or Fog 1 mRNA levels as compared tothe levels observed with pre-treated mesenchymal stem cells. Thesedifferentiated cardioprogenitor cells also exhibited the ability toincorporate into heart tissue as functional cardiomyocytes when injectedintramyocardially, subcutaneously, or intravascularly with heart pumpfunction improvement directly correlated with structural repair in bothischemic and non-ischemic settings. Functional benefit was documentedboth echocardiographically in vivo, and histologically on autopsythrough staining of human specific proteins. Also as described herein,differentiated cardioprogenitor cells derived from mesenchymal stemcells cultured with serum containing TGFβ-1, BMP4, FGF-2, IGF-1,Activin-A, Cardiotrophin, α-thrombin, and Cardiogenol C exhibited a 5 to10-fold increase in MEF2c mRNA, MESP-1 mRNA, and Tbx-5 mRNA levels ascompared to the levels observed with pre-treated mesenchymal stem cells.

These differentiated cardioprogenitor cells also exhibited the abilityto incorporate into heart tissue as functional cardiomyocytes wheninjected intramyocardially (e.g., through endocardial or epicardialroutes), into the coronary arteries, infused in the heart, oradministered systemically (e.g., subcutaneously), with heart pumpfunction improvement directly correlated with structural repair in bothischemic and non-ischemic settings. Functional benefit was documented bycardiac ultrasound in vivo, and by microscopic analysis on autopsythrough staining of human specific proteins. Thus, release criteria suchas elevated polypeptide or mRNA levels of MEF2c, MESP-1, Tbx-5, GATA6,Flk-1, Fog 1, FOG 2, or combinations thereof can be used to evaluatecells prior to administration into a mammal.

The term “elevated level” as used herein with respect to polypeptide ormRNA levels of MEF2c, MESP-1, Tbx-5, GATA6, Flk-1, or Fog (for instanceFOG 1 for mRNA, FOG 2 for the polypeptide) within a cell populationrefers to any level that is greater than a reference level for thatpolypeptide or mRNA.

The term “reference level” as used herein with respect to polypeptide ormRNA levels of MEF2c, MESP-1, Tbx-5, GATA6, Flk-1, or Fog (for instanceFOG 1 for mRNA, FOG 2 for the polypeptide) within a cell populationrefers to the level typically found in pre-treated cells (e.g.,pre-treated mesenchymal stem cells). For example, an MEF2c mRNAreference level, an MESP-1 mRNA reference level, a Tbx-5 mRNA referencelevel, a GATA6 mRNA reference level, and a FOG 1 mRNA reference levelcan be the average level of MEF2c, MESP-1, Tbx-5, GATA6, Flk-1 and FOG 1mRNA, respectively, that is present in a random sampling of mesenchymalstem cells not treated with a composition provided herein or otherwisetreated with differentiation factors. It will be appreciated that levelsfrom comparable samples are used when determining whether or not aparticular level is an elevated level.

Elevated polypeptide and/or mRNA levels of MEF2c, MESP-1, Tbx-5, GATA 4,GATA6, Flk-1, Fog 2 or FOG 1 can be any level provided that the level isgreater than a corresponding reference level.

For example, an elevated level of Tbx-5 mRNA can be 1.5, 2, 3, 4, 5, 6,7, 8, 9, 10, or more fold greater than the reference level Tbx-5 mRNAobserved in untreated mesenchymal stem cells. It is noted that areference level can be any amount. For example, a reference level forTbx-5 mRNA can be zero. In this case, any level of Tbx-5 mRNA greaterthan zero would be an elevated level.

In some cases, identification criteria can include microscopic analysisof cells prior to administration into a mammal. Such microscopicanalysis can include assessing the cells for transcription factorpolypeptides associated with the nucleus. For example, cells appropriatefor release into a mammal can be assessed for the presence of Nkx2.5,MEF2c, GATA4, MESP-1, FOG 2, Tbx-5, or any combination thereofassociated with the nucleus before being released into the mammal.

Any appropriate method can be used to provide heart tissue withdifferentiated cardioprogenitor cells having the ability to incorporateinto heart tissue as functional cardiomyocytes. For example,differentiated cardioprogenitor cells can be injected intramyocardially(e.g., through endocardial or epicardial routes), into the coronaryarteries, infused in the heart, or administered systemically (e.g.,subcutaneously).

Any heart tissue can be provided with differentiated cardioprogenitorcells. For example, mammalian (e.g., human) heart tissue can be providedwith differentiated cardioprogenitor cells. In some cases, heart tissuethat has suffered from ischemic cardiomyopathy, myocardial infarction,or heart failure can be provided with differentiated cardioprogenitorcells.

Any type of differentiated cardioprogenitor cells can be administered toheart tissue. For example, autologous or heterologous differentiatedcardioprogenitor cells can be administered to heart tissue. In somecases, stem cells (e.g., mesenchymal stem cells) that were incubatedwith a composition provided herein can be administered to heart tissue.

The stem cells can be incubated with a composition provided herein forany length of time before being administered to heart tissue. Forexample, the stem cells can be incubated with a composition providedherein for 6 to 24 hours (e.g., 8, 10, 12, 18, or 22 hours) or for 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40, 45, or 50 days before being administered toheart tissue. In some cases, stem cells that were incubated with acomposition provided herein can be administered to heart tissue togetherwith a composition provided herein.

The stem cells can be incubated with a composition provided herein forany length of time before being administered to heart tissue togetherwith a composition provided herein. For example, the stem cells can beincubated with a composition provided herein for 6 to 24 hours (e.g., 8,10, 12, 18, or 22 hours) or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or50 days before being administered to heart tissue together with acomposition provided herein.

In some cases, differentiated cardioprogenitor cells can be assessed todetermine whether or not they meet particular release criteria prior tobeing administered to a mammal. For example, differentiatedcardioprogenitor cells can be assessed using RT-PCR to confirm that thedifferentiated cardioprogenitor cells express an elevated polypeptide ormRNA level of MEF2c, MESP-1, Tbx-5, GATA6, Flk-1, Fog, (FOG 1 for mRNA,FOG 2 for the polypeptide) or combinations thereof before beingadministered into a mammal.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the change of ejection fraction (ΔEF) in %, consideredbefore and after treatment with naïve human mesenchymal stem cells(hMSCs) derived from bone narrow of 11 patients with coronary arterydisease.

FIG. 2 obtained on confocal microscopy after immunostaining with DAPI,shows protein expression of the cardiac transcription factor content forpatient 2 that demonstrated no positive ejection fraction change (left)and for patient 9 (right) that demonstrated a positive ejection fractionchange after treatment.

FIG. 3 shows the mRNA expression of two significant cardiactranscription factors mRNA in the hMSCs of the eleven patients.

FIG. 4 shows the mRNA expression, in arbitrary units (A.U.) of cardiactranscription factors for respectively Nkx2.5 mRNA, GATA-6 mRNA andFog-1 mRNA, of untreated, naïve hMSCs (left) and on CP-hMSCs, treatedwith a cardiogenic cocktail (right).

FIG. 5 shows images obtained on confocal microscopy showing nucleartranslocation of Nkx2.5, MEF2C, FOG-2 and GATA4 polypeptides in CP-hMSCstreated with a cardiogenic cocktail (right), compared with naïve hMSCs(left).

FIG. 6 illustrates the progressive conversion (at days D0, D5, D15, andday D20) from naïve hMSCs to ‘cocktail-guided’ CP-hMSCs and eventuallycardiomyocytes (CM).

FIG. 7 shows the transition electron microscopy ultra structure of naïvehMSC and cocktail-guided cardiomyocyte.

FIG. 8 shows a cocktail-guided cardiomyocyte in light microscopy.

FIG. 9 represents, on the first graph on the left, the Tbx-5 mRNAexpression (in AU) for naïve hMSC and CP-hMSC from each patient of FIG.1, the results for the naïve cells being on the right and the one forthe CP cells being on the left of the histogram. The second graph in themiddle presents MEF2C mRNA expression, and the third graph on the rightpresents MESP-1 mRNA expression. The average values for the naïve cellsfrom all patients and the one for the CP cells for all patients aregiven in the background of the histogram.

FIG. 10 is a graph which represents the ejection fraction change(AEjection Fraction) after treatment of heart with different quantitiesof naïve hMSC (right part of histogram for each of twelve patients)versus treatment of hearts with CP-hMSCs, i.e. cocktail-guided hMSC(CP-hMSC).

FIG. 11 represents the echocardiography of infarcted hearts untreated(left) and treated with cardiopoietic cells (right), which shows a farbetter anterior wall reanimation upon treatment with CP-hMSCs.

FIG. 12 is a graph similar to FIG. 5 showing the ejection fractionchange (ΔEF) after injection of naïve (on the left) and cardiopoieticcells (on the right) into infracted myocardium. Sham is injectionwithout cells.

FIG. 13 shows murine infarcted hearts treated with naïve hMSC andCP-hMSCs six months following treatment initiation. Aneurysms and scar,which remained uncorrected in naïve hMSCs treated hearts, were resolvedwith CP-hMSCs treatment that induced re-muscularization.

FIG. 14 shows that confocal resolution revealed, in the cardiopoietichMSC treated murine myocardium, widespread presence of human-derivedcells with positive staining for h-ALU-DNA sequences specific to thehuman species validated with human-specific lamin immunostaining, all ofwhich were absent in infarcted controls.

FIG. 15 shows that cardiomyocytes of human origin were tracked byco-localization of human cardiac troponin-I and α-actinin incardiopoietic hMSC-treated hearts. Cardiomyocytes of human origin wereabsent from naïve hMSC-treated hearts. Quantification within infarctedanterior walls revealed 3±2% and 25±5% of myocardial nuclei in naïveversus CP-hMSC-treated hearts, implying enhanced engraftment withcardiopoietic hMSC treatment.

FIG. 16 contains photographs of naïve and CP hMSC stained for humantroponin, ventricular myosin light chain mIC2V, and DAPI. Ventricularcell phenotype was corroborated with counter staining of human-troponinpositive cells with ventricular myosin light chain mIC2V immunostainingin repaired anterior wall, as shown in FIG. 15 or resolving scar asshown in FIG. 17.

FIG. 17 is a photograph of a remnant scar stained for human troponin,ventricular myosin light chain mIC2V, and DAPI.

FIG. 18 contains photographs of CP-hMSCs-treated regenerating myocardiumdemonstrating angiogenesis distal to the occluded coronary vessel.

FIG. 19 demonstrates CP-hMSCs contribution to neo-vascularization viaexpression of human PECAM-1 (CD-31) within the myocardial vasculature.

FIG. 20 is a graph plotting Δ ejection fraction relative to sham (%)versus time (months) following cell delivery. The long-term impact ofCP-hMSCs treatment was tracked for more than one 1-year, or one third ofmurine lifespan which would translate into 25-years of human life.Relative to sham, treatment with naïve hMSC showed a 5% and 2.5%ejection fraction effect at 6 and 12 months, respectively. In contrast,cardiopoietic hMSC treated infarcted mice demonstrated significantejection fraction improvement of 25% at 6 and 12 months relative tosham.

FIG. 21 is a graph plotting Δ ejection fraction (%) versus time (months)post cell transplantation. The infarcted cohort was stratified toevaluate efficacy in subgroups with documented overt heart failure(ejection fraction <45%) at the time of intervention. Despite equivalentpre-treatment ejection fraction at 35%, only cardiopoietic hMSCtreatment improved absolute ejection fraction by 10% at 6 and 12-months,in contrast to a 5% decline in ejection fraction in the naïvehMSC-treated cohort.

FIG. 22 is a bar graph plotting survivorship (%) for the indicatedsubgroups of mice. In overt heart failure subgroups at 400 daysfollow-up, no survivors were present in the sham and mortality of >50%was recorded with naïve hMSC treatment. In contrast, a >80% survival wasattained with cardiopoietic hMSC treatment.

FIG. 23 illustrates the safety of treatment with CP-hMSCs, determined bypathological examination and electrocardiography.

EXAMPLES

Patients undergoing coronary artery bypass for ischemic heart diseasewere randomly selected for bone marrow harvest. They provided informedconsent, and study protocols were approved by pertinent InstitutionalEthics Committee and Institutional Animal Care and Use Committee. Isworth noting that no injections were made to patients but to mice.

Example 1

Mesenchymal stem cells were derived from human bone marrow withdrawnfrom the posterior iliac crest of the pelvic bone of 18- to 45-year-oldhealthy individuals (Cambrex, East Rutherford, N.J.). Based on flowcytometry analysis, the mesenchymal stem cells expressed CD90, CD133,CD105, CD166, CD29, and CD44, and did not express CD14, CD34, and CD45.

Human bone marrow-derived mesenchymal stem cells were cultured in eitherplatelet lysate or serum supplemented with TGFβ-1 (2.5 ng/ml), BMP4 (5ng/ml), FGF-2 (5 ng/ml), IGF-1 (50 ng/ml), Activin-A (10 ng/ml),Cardiotrophin (1 ng/ml), α-thrombin (1 Unit/ml), and Cardiogenol C (100nM). After 4-10 days in the platelet lysate-containing culture at adensity of about 1000-2000 cells per cm², the cells were found toexpress 2-5-fold more MEF2c mRNA, MESP-1 mRNA, Tbx-5 mRNA, GATA6 mRNA,Flk-1 or FOG 1 mRNA than untreated mesenchymal stem cells.

After 5-15 days in the serum-containing culture at a density of about1000-2000 cells per cm², the cells were found to express 5-10-fold moreMEF2c mRNA, MESP-1 mRNA, Tbx-5 mRNA, GATA 4 mRNA, GATA6 mRNA, Flk-1 orFOG 1 mRNA than untreated mesenchymal stem cells.

The primer pairs used for the RT-PCR analysis were standard primersobtained commercially from Applied Biosystems.

Results demonstrating that the differentiated cardioprogenitor cellshave the ability to incorporate into heart tissue as functionalcardiomyocytes were obtained both in vivo within the beating heart, andin vitro following autopsy. In vivo, under isoflurane anesthesia, directmyocardial delivery of cardioprogenitor cells into diseased heartsimproved cardiac performance as monitored by echocardiography in theshort axis with a two-dimensional M-mode probing in the long axis,Doppler pulse wave analysis, and 12-lead electrocardiography.

Harvested heart tissue was fixed in 3% paraformaldehyde, sectioned, andsubjected to immuno-probing for human cell tracking. New human derivedcardiomyocytes and vasculature, with functional improvement and scarresolution, was documented on analysis in mice treated withcardioprogenitor cells fulfilling release criteria (e.g., elevated levelof expression of MEF2c mRNA, MESP-1 mRNA, Tbx-5 mRNA, GATA 4 mRNA, GATA6mRNA, Flk-1 or FOG 1 mRNA), in contrast to absence of benefit with cellsthat did not pass the release criteria.

In order to scale-up the production of cardiopoietic cells forautologous injection in patients, an alternative method was consideredas immunofluorescence can be time-consuming, qualitative and potentiallyoperator-dependent. One method of choice is real-time quantitativereverse transcription polymerase chain reaction (RT-qPCR). This methodgives faster results (within one day) that are operator-independent andquantified relative to a reference standard. In addition, whileimmunostained samples require one by one fluorescent microscopyevaluation, up to 48 different samples (or conditions) can be tested induplicate by RT-qPCR using 96-well plates.

In order to identify suitable markers for RT-QPCR, cardiopoietic cellswere derived from bone marrow samples obtained from cardiac patients(n=7). Cells were evaluated by immunofluorescence staining for MEF2C andNkx2.5. RNA was extracted from these cells and expression of Nkx2.5 andMEF2C measured by real-time quantitative PCR.

The reference standard consisted of cells from the same batch notcultured in the presence of the cardiogenic cocktail.

Results were calculated using the double delta-Ct method normalizing thedata obtained from treated cells to those from untreated cells.

MEF2C was identified as suitable marker of cardiopoietic cells by bothqPCR and immunofluorescence (nuclear translocation) when compared tonaive cells. By contrast, the qualitative change in Nkx2.5 seen at theprotein level by immunofluorescence (nuclear translocation) wasinitially not translated into a quantitative change at the RNA levelrelative to untreated cells. Genes downstream of Nkx2.5 were theninvestigated, since induction of their expression would depend onnuclear translocation of Nxk2.5. This led to the identification ofMESP-1, Flk-1 and Tbx5 as additional suitable genes for identificationby QPCR.

Human bone marrow aspirates (15-20 ml) were obtained during coronaryartery bypass surgery following sternotomy. Bone marrow was cryostoredin a DMSO-based serum-free freezing solution. Mesenchymal stem cellswere recruited by platting of raw bone marrow on plastic dishes with awash at 12 h selecting adhesive cells with identity confirmed byFluorescence-Activated Cell Sorting (FACS) analysis using theCD34⁻/CD45⁻/CD133⁺ marker panel. Cells were cultured at 37° C. in DMEMsupplemented with 5% human platelet lysate (Mayo Clinic Blood Bank,Rochester, Minn.).

Myocardial infarction was performed in nude, immunocompromised mice(Harlan, Indianapolis, Ind.). Following a blinded design, one monthpost-infarction a total of 600,000 naïve or cardiopoiesis guided hMSC,suspended in 12.5 μl of propagation medium, was injected undermicroscopic visualization in five epicardial sites on the anterior wallof the left ventricle. Sham underwent the same surgical procedurewithout cell injection. Myocardial injection of bone marrow hMSC intothis chronic infarction model demonstrated heterogeneity in outcome withtransplantation of cells from only two out of the eleven studiedindividuals improving ejection fraction on echocardiography.

Patients 3 and 9 were identified as individuals with a highcardio-generative potential. It was first observed from FIG. 1 that thechange of ejection fraction in mice (n=3) treated with hMSC from eachpatients 3 and 9 was significantly positive, whereas the change for eachother patient was not.

The protein expression of cardiac transcription factors was observed inhMSC on confocal microscopy, as shown in FIG. 2. Bar corresponds to 20μm representative for all panels.

Immunostaining was performed with antibodies specific for MEF2C (1:400,Cell Signaling Technologies, Danvers, Mass.), Nkx2.5 (1:150, Santa CruzBiotechnology Inc., Santa Cruz, Calif.), GATA4 (Santa Cruz BiotechnologyInc.), Phospho-AKT^(Ser473) (1:100, Cell Signaling Technologies), Tbx5(1:5000, Abcam, Cambridge, Mass.), Mesp-1 (1:250, Novus Bio, Littleton,Colo.), Fog-2 (1:100, Santa Cruz Biotechnology), sarcomeric proteinα-actinin (1:500, Sigma-Aldrich) and human-specific Troponin-I (1:100,Abcam), mIC2v (1:500, Synaptic Systems, Gottigen, Germany), Sca-1(1:100, R&D Systems, Minneapolis, Minn.), CD-31/PECAM-1 (1:500, BeckmanCoulter, Fullerton, Calif.), α-smooth muscle actin (Abcam),human-specific Troponin-I (1:100, Abcam), human Lamin A/C (1:50,Novacastra, New Castle, UK), and Ki67 (1:500, Abcam) following fixationin 3% paraformaldehyde and permeabilization with 1% Triton X-100, andalong with DAPI staining to visualize nuclei on confocal microscopyperformed with a LSM 510 Laser scanning confocal microscope (Carl ZeissInc., Jena, Germany).

Early cardiac transcription factors Nkx 2.5, Tbx-5 and MESP1 latecardiac transcription factor MEF2C were observed under staining withDAPI. The results for patient 2 are on the left, the one for patient 9on the right. The images obtained show that the expression of thecardiac transcription factors is weak for the hMSC from patient 2 andhigh for the one of patient 9. This corroborates the fact that the hMSCfrom patient 9 give an efficient therapeutic benefit whereas the hMSCfrom patient 2 do not. The coloration afforded by DAPI is blue.

On FIG. 2 the first series of images for Nkx 2.5 show the nuclei of thecells colored DAPI (left) solely blue for the hMSC of patient 2 (left).A weak green colouration corresponding to the presence of Nkx 2.5 in thecytoplasm also appears. The corresponding image for patient 9 (right)shows a higher expression of Nkx 2.5 (green) in the cytoplasm and alsoin the nuclei of the cells.

The second series of images show the cardiac transcription factors Tbx-5(green) and MESP-1 (red) for patient 2, the nuclei of the cells andcoloured in blue by the DAPI, no green or red colour is visible, whichcorresponds to no expression of TbX-5 and MESP-1. For patient 2, thecytoplasms of the cells are coloured in red and the nuclei in green,which corresponds to strong expression of both cardiac transcriptionfactors and to a translocation of Tbx-5 to the nuclei of the cells.

The third series of images gives results for MEF2C similar to the onefor Nkx 2.5.

FIG. 3 shows the mRNA expression studied in qPCR revealing cardiactranscription factor expression (MEF2C and Tbx-5) for the hMSC of theeleven patients of the study.

Quantitative polymerase chain reaction (qPCR) was performed using aTaqMan PCR kit with an Applied Biosystems 7,900HT Sequence DetectionSystem (Applied Biosystems, Foster City, Calif.). TaqMan Gene Expressionreactions were incubated in a 96-well plate and run in triplicate. Thethreshold cycle (C_(T)) was defined as the fractional cycle number atwhich fluorescence passes a fixed threshold. TaqMan C_(T) values wereconverted into relative fold changes determined using the 2^(−ΔΔC) ^(T)method, normalized to GAPDH (P/N 435,2662-0506003) expression.

Genes listed in Table 1, which are representative of cardiactranscriptional activity were evaluated.

Cells were evaluated at the mRNA and protein levels prior to andfollowing a 5-day stimulation with a cardiogenic cocktail comprisinghuman recombinant TGFβ-1 (2.5 ng/ml), BMP4 (5 ng/ml), Cardiotrophin (1ng/ml), α-thrombin (1 U/ml), and Cardiogenol C (100 nM). Both the MEF2CmRNA and the Tbx-5 mRNA expressions (in arbitrary units AU) are muchhigher for the hMSC of patients 2 and 9 than for the one of otherpatients.

TABLE 1 Applied Biosystems Assay ID Gene name Gene symbol Hs00231763_m1Homeobox transcription factor or Nkx2.5 or NKX2-5 NK2 transcriptionfactor related, locus 5 or NKX2.5 Hs00171403_m1 zinc finger cardiactranscription factor or GATA-4 or, GATA binding protein 4 GATA4 (AB)Hs00231149_m1 myocyte enhancer factor 2C MEF2c or MEF2C Hs00361155_m1T-box transcription factor or Tbx5 or TBX5 T-box 5 Hs00542350_m1 GATAco-factor (“Friend of GATA”) or FOG 1 of FOG-1 zinc finger protein,multitype 1 or FOG1 Hs00251489_m1 Helix-loop-helix transcription factorMesp1 or MESP1 mesoderm posterior 1 homolog (mouse) (AB) Hs00232018_m1GATA binding protein 6 (AB) GATA-6 or GATA6 Hs00911699_m1 Kinase insertdomain receptor (a type Flk-1, or III receptor tyrosine kinase) FLK1 orKDR

Left ventricular function and structure were serially followed bytransthoracic echocardiography (Sequoia 512; Siemens, Malvern, Pa. andVisualSonics Inc, Toronto, Canada). Ejection fraction (%) was calculatedas [(LVVd−LVVs)/LVVd]×100, where LVVd is left ventricular end-diastolicvolume (μl), and LVVs is left ventricular end-systolic volume (μl).

FIG. 4 shows the mRNA expression, in arbitrary units (A.U.) of cardiactranscription factors for respectively Nkx2.5 mRNA, GATA-6 mRNA andFog-1 mRNA, of untreated, naïve hMSC (left) and on CP-hMSC, treated witha cardiogenic cocktail (right). It is clear, in each case, that theresults are far better when using cells treated with a cardiogeniccocktail.

FIG. 5 shows images obtained on confocal microscopy showing nucleartranslocation Nkx2.5, MEF2c, GATA4 and FOG-2 polypeptides in naïveCP-hMSC treated with a cardiogenic cocktail. Nkx2.5, MEF2c, GATA4 andFOG-2 appear in green and DAPI in blue. On the images of naïve hMSCs, notranscription factor appears. The polypeptides are translocated on thenuclei of CP-hMSCs (right) as indicated by the concentrated greencolour.

FIG. 6 illustrates the progressive conversion (at days D0, D5, D15, andday D20) from naïve hMSCs to ‘cocktail-guided’ CP-hMSCs and eventuallycardiomyocytes, CM. On D0, nuclei are coloured in blue by DAPI. On D5,MEF2C polypeptide is translocated on nuclei (green). On D15, sarcomericα-actinin is present (red), which shows that sarcomeres are present andhence that the cells are definitively engaged into the cardiomyocyticdifferentiation and are no longer cardiopoietic. A large quantity oftroponin-1 is present in cardiomyocytes on D20 (terminaldifferentiation).

FIG. 7 shows the transition electron microscopy ultrastructure of naïvehMSC (left) and cocktail-guided cardiomyocyte (right). To this end,cells were cultured in 1% platelet lysate for 15 days The cardiomyocytespresent a mitochondrial maturation, a sarcomerogenesis and formation ofmyotubes.

FIG. 8 shows a cocktail-guided cardiomyocyte in light microscopy.Maturation of the excitation-contraction system was assessed throughinduction of calcium transients. To this end, cells were cultured for 15days following 5 days of cocktail stimulation and loaded for 30 min at37° C. with 5 μM of the calcium-selective probe fluo-4-acetoxymethylester (Molecular Probes, Carlsbad, Calif.) for live imaging using atemperature controlled Zeiss LSM 510 microscope (Zeiss) and line-scanimages acquired during 1 Hz stimulation.

FIG. 9 shows a 3-, 8-, and 8-fold increase in Tbx-5, MEF2C and MESP-1 intreated versus untreated hMSC.

As shown in FIG. 10, CP-hMSCs meeting CARPI criteria, were delivered invivo one-month following infarction and significantly improved ejectionfraction over naïve patient-matched hMSC.

FIG. 11 represents an echocardiography of infarcted hearts untreated(left) and treated with cardiopoietic cells (right), which shows a farbetter anterior wall reanimation upon treatment with CP-hMSCs.Electrocardiograms were recorded using four-limb-leadelectrocardiography (MP150; Biopac, Goleta, Calif.) under lightanaesthesia (1.5% isoflurane).

On echocardiography, contractility improved by 15% and 20% at one- andtwo-months, respectively, following treatment with CP-hMSC (n=14), incontrast to marginal change recorded with naïve hMSC (n=17) or sham(n=10; FIG. 9). On top: Echocardiography of infarcted hearts 4 weeksfollowing coronary ligation and 1 day prior to cellular transplant (4wks post MI—no Tx) revealed on M-Mode an akinetic anterior wall in bothstudy groups. Middle: 4 weeks after cellular transplantation (4 wks postCell Tx), naïve hMSC treated hearts demonstrated maintained akinesis inthe anterior wall, in contrast to re-animation in the CP-hMSCs treatedgroup. Lower: 8 weeks following cellular transplantation (8 wks postCell Tx), naïve hMSC treated hearts continued to show limited evidencefor myocardial repair versus robust contractile activity in the CP hMSCtreated infarcted hearts. Left panels represent para-sternal (PS) longaxis views, with dash line indicating level of 2-D M-Mode capture.Arrowheads in right panels indicate anterior wall re-animation.

FIG. 12 shows that on average, guided cardiopoietic hMSC achieved amarked improvement at the one and two month follow-up followinginjection into infarcted myocardium. In contrast, naïve hMSC or shamcontrols had limited impact on ejection fraction. Star and double starindicate a p<0.01 over naïve hMSC for the two time points.

In hearts treated with cardiopoietic cells derived from hMSC, functionalimprovement correlated on three-month and 18-month histopathologicalevaluation with myocardial regeneration. Aneurysms and scar, whichremained uncorrected in naïve hMSC-treated hearts, were resolved withcardiopoietic hMSC treatment that induced re-muscularization (FIG. 13).

Gross pathological evaluation demonstrated resolution of scar downstreamof left anterior descending (LAD) artery ligation (yellow circle on thehearts) with, on cross-section, robust re-muscularization and diminishedremodeling in cardiopoietic (CP, right) in contrast to naïve (left)hMSC-treated infarcted hearts at 6-months following treatmentinitiation. These results are particularly good.

Probing for ALU-DNA was performed using human ALU-Probe (Biogenex, SanRamon, Calif.) by hybridization at 85° C. for 5-10 minutes andincubation at 37° C. overnight followed by anti-Fluorescein GFP-labeledsecondary detection.

Confocal resolution revealed, in the CP-hMSC-treated murine myocardium,widespread presence of human-derived cells with positive staining forALU DNA sequences specific to the human species validated withhuman-specific lamin immunostaining, all absent in infarcted controls(FIG. 14).

In contrast to sham (left), cardiopoietic hMSC treated hearts onconfocal microscopy evaluation revealed dramatic presence of humannuclei as stained by a human h-ALU DNA probe (middle) imbedded withinthe murine infarcted myocardium, further confirmed with additionalstaining for a human-specific lamin antibody (right, inset shown in FIG.14). Frozen myocardial sections were made from super-oxygenated 3%paraformaldehyde in PBS perfusion fixed hearts. Bar indicates 50 μm.

FIG. 15 shows that human-specific troponin-I antibody revealed nostaining in naïve (left) versus significant staining in the anteriorwall of cardiopoietic hMSC treated hearts (middle and right panels)

Moreover, as shown in FIG. 16 human troponin-I staining of naïve (top)versus cardiopoietic (bottom) hMSC treated hearts, counterstained withmIC2v, demonstrated generation of ventricular myocardium from engraftedhuman cells. Bars indicate 20 μm (top) and 50 μm (bottom).

As illustrated in FIG. 17 within the remnant scar of cardiopoieticderived from hMSC-treated hearts, human stem cell derived myocardiumcould be distinguished from native murine myocardium with human troponincolocalization with mIC2V. Bar indicates 50 μm.

In FIG. 18 surface microscopy detected angiogenesis distal to theligated LAD (black circle) in CP-hMSCs-treated hearts arising from theright coronary artery (RCA; left bottom) and circumflex (right bottom).

FIG. 19 shows confocal evaluation of collateral vessels fromcardiopoietic hMSC treated hearts demonstrated human-specific CD-31(PECAM-1) staining. Bar represents 20 μm.

FIG. 20 shows the evolution of the change of ejection fraction relativeto sham in %, during 12 months, for treatment with both naïve andcocktail-guided (CP) hMSC. Relative to sham, treatment with naïve hMSCshowed a 5% and 2.5% ejection fraction effect at 6 and 12 months,respectively.

In contrast, CP-hMSCs-treated infarcted mice demonstrated significantejection fraction improvement of 25% at 6 and 12 months relative to sham(FIG. 20). Furthermore, the infarcted cohort was stratified to evaluateefficacy in subgroups with documented overt heart failure (ejectionfraction <45%) at the time of intervention. Despite equivalentpre-treatment ejection fraction at 35%, only cardiopoietic hMSCtreatment improved absolute ejection fraction by 10% at 6 and 12-months,in contrast to a 5% decline in ejection fraction in the naïvehMSC-treated cohort (FIG. 23). As shown in FIG. 22, superior survivalbenefit in the cardiopoietic hMSC treated group in contrast to naïvetreated cohort and sham was determined through application of theKaplan-Meier function with censoring.

Efficacy of cardiopoietic (CP) hMSC was demonstrated by echocardiographyat 1-year follow-up (see FIG. 25). Long axis imaging of naïve stem celltreated hearts revealed a fibrotic and hypokinetic anterior wall mostevident on apical M-Mode evaluation (Patient 11, left panels). Incontrast, CP-hMSC-treated hearts revealed a robust contractile profilethroughout the anterior wall reflecting a sustained benefit from guidedstem cell therapy (Patient 11, right panels).

Example 2

Similar results have been observed by treating stem cells with acocktail containing recombinant TGFβ-1 (2.5 ng/ml), BMP4 (5 ng/ml),Cardiotrophin (1 ng/ml), Cardiogenol C (100 nM) and α-thrombin, (1U/ml), FGF-2 (10 ng/ml), IGF-1 (50 ng/ml) and Activin-A (5 ng/ml) usedin a combinatorial fashion.

Example 3

Similar results have been observed by treating stem cells with acocktail containing recombinant TGF-β1 (2.5 ng/ml), BMP-4 (5 ng/ml),Activin-A (5 ng/ml), FGF-2 (10 ng/ml), IL-6 (100 ng/ml), Factor IIa(hα-thrombin, 1 U/ml), IGF-1 (50 ng/ml), and retinoic acid (1 μM) usedin a combinatorial fashion.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

The invention claimed is:
 1. A composition for use in differentiation ofstem cells into cardioprogenitor cells consisting of: TGFβ-1; a BMPpolypeptide, wherein the BMP polypeptide is selected from the groupconsisting of: BMP2 and BMP4; α-thrombin; FGF-2; IGF-1; Activin-A;Cardiotrophin; and Cardiogenol C.
 2. The composition of claim 1, whereinthe BMP polypeptide is BMP4.
 3. The composition of claim 1, wherein whenone compound is present in said composition, it is present in an amountof between 1 and 5 ng of said TGFβ-1 per mL, between 1 and 10 ng of saidBMP4 per mL, between 0.5 and 5 ng of said Cardiotrophin per mL, between0.5 and 5 units of said α-thrombin per mL, between 50 and 500 nM of saidCardiogenol C, between 1 and 10 ng of said FGF-2 per mL, between 10 and100 ng of said IGF-1 per mL, and between 1 and 50 ng of said Activin-Aper mL.
 4. The composition of claim 1, containing 2.5 ng/mL ofrecombinant TGFβ-1, 5 ng/mL of BMP4, 1 ng/mL of Cardiotrophin, and 100nM of Cardiogenol C.
 5. The composition of claim 4, further comprising 1U/mL of α-thrombin, 10 ng/mL of FGF-2, 50 ng/mL of IGF-1, and 5 ng/mL ofActivin-A.
 6. The composition of claim 1, which is comprised in a mediumselected from the group consisting of media containing fetal calf serum,media containing human serum, media containing platelet lysate, andmedia containing mixtures thereof.